Purification And Anti-cancer Activities Of Several Natural Products And Their Underlying Mechanisms

Malignancy, one of the harmful diseases, accounts for 12.5% of human deaths every year worldwide, and chemotherapy still plays an important role in the treatment of cancer. However, most of chemotherapy agents possess serious side effects. Therefore, much attention has been paid to find novel anticancer agents with low toxicity.Halophilic microorganisms possess the unique metabolic and physiological capabilities. Therefore, there is a potential to produce unique metabolites from halophilic microorganisms. In the present study, two moderately halophilic fungi F1 and F2 were isolated from a marine solar saltern in Weihai, China. The identification of the fungi were performed by the morphological characteristics, physiological and biochemical tests as well as phylogenetic analysis based on ITS(internal transcribed spacer)-5.8S rDNA region sequence comparison. The strain F1 was identified as belonging to a new species of the genus Aspergillus and designated as Aspergillus sp. nov. F1. The strain F2 was identified as belonging to the genus Penicillium and designated as Penicillium chrysogenum F2. Furthermore, Aspergillus sp. nov. F1 grew well in 3-15 %(w/v) NaCl, and with increasing of salinity, the generation of secondary metabolites with cytotoxicity was also augmented.The active components with cytotoxicity were purified by silica gel, Sephadex LH-20 columns and recrystallization and identified using 1H-NMR, 13C-NMR and ESI-MS. Three compounds with cytotoxicity were isolated from the ethyl acetate extract of the whole broth and mycelia of Aspergillus sp. nov. F1, and identified as ergosterol, rosellichalasin and cytochalasin E, respectively. Two compounds with cytotoxicity were isolated from the ethyl acetate extract of the whole broth and mycelia of Penicillium chrysogenum F2, and identified as ergosterol and meleagrin, respectively. Ergosterol showed high potent cytotoxic activity to human colon cancer RKO cell line with IC50 of 3.3±0.5 μM. Considering the high cytochalasin production and the simple and economical fermentation of Aspergillus sp. nov. F1, the strain could be used as potential strain for large scale production of the cytochalasin E and rosellichalasin.Ergosterol as a predominant sterol component found in the plasma membrane of fungus, is a pentacyclic triterpenoid. Our study showed that freshly prepared ergosterol displayed low cytotoxicity on inhibiting the proliferation of HeLa cells. However, ergosterol derivatives(ED) showed high potent cytotoxic activity to a wide range of human tumor cell lines; the IC50 of ED was 9.1±1.64 μg/mL, as determined by MTT assay. Further study showed that ED could induce remarkable apoptosis, evidenced by Hoechst 33258 staining assay. Treatment of the cancer cells with ED resulted in inhibition of NF-κB p65 activation and nuclear translocation. The expression of NF-κB-dependent reporter genes Bcl-2, Survivin, COX-2 and VEGF was also significantly attenuated. The cell cycle was arrested in S phase of HeLa cells in the presence of ED. In addition, ED caused the disassembly of the microtubule cytoskeleton in HeLa cells. These studies suggested that ED induced cell death and apoptosis via NF-κB signaling pathway in HeLa cells. These studies provide evidence that ergosterol derivatives have the potential to become novel anticancer agents with low side effects.The anticancer activity of cardamonin was also studied in this study. Cardamonin, a naturally occurring chalcone, possess anti-inflammatory and anti-tumor activity against many tumor cell lines. However, little is known about their mode of action. This study aims to study the anti-tumor activity of cardamonin on human colon cancer cells and its potential molecular mechanism. The results of MTT assay showed that the proliferation of RKO cells was inhibited by cardamonin in a concentration-dependent manner, with an IC50 of 4.25 μg/mL. Cardamonin could induce remarkable apoptosis, evidenced by an increased percentage of apoptotic cells, DNA ladder and nuclear morphology change. Further study revealed that cardamonin could induce generation of Reactive Oxygen Species(ROS), mitochondrial depolarization, release of cytochrome C via mitochondrial pathway. The expression of apoptosis-correlated proteins was also affected in cells treated with cardamonin, including activation of caspases-3/9 and cleavage of PARP. Furthermore, the cell cycle was arrested in S phase, and the activity of topoisomerase I was also inhibited in the presence of cardamonin. The study provides insight into the understanding of anticancer mechanism of cardamonin.